The absence of experimental and computational tools for global identification of phosphatase substrates leaves a major gap in our understanding of cellular regulatory networks and prevents systems-level analyses of phosphatase signaling. The proposal addresses this knowledge gap by systematically identifying targets of calcineurin (CN), the ubiquitous Ca2+/calmodulin-dependent protein phosphatase and target of immunosuppressants, FK506 and cyclosporin A. Only 25 substrates are currently attributed to CN in mammals--the same as in yeast, whose proteome is ~tenfold smaller, and chronic inhibition of CN in patient's cause's side-effects by disrupting unidentified regulatory events. This suggests that in humans, the majority of CN substrates, and thus its regulatory functions, remain to be identified. Recent insights into CN substrate recognition drive our approach: CN binds to short linear motifs (SLiMs), termed PxIxIT and LxVP, which can occur hundreds of residues away substrate dephosphorylation sites. Mutating these motifs or preventing their binding to conserved surfaces on CN, i.e. with FK506, CysA, or viral inhibitor A238L, blocks dephosphorylation. Building upon our previous success establishing the CN signaling network in yeast, we are determining human CN substrates by systematically identifying CN- binding peptides in the proteome, which is challenging due to their low affinities and degenerate sequences. This work aims to 1) systematically discover human PxIxIT and LxVP-type sequences by synergizing experimental and computational methods. CN-binding sequences are directly selected from phage display libraries that contain all disordered regions of the human proteome, where SLiMs reside. PxIxITs are also identified in silico by leveraging their characteristic structural features and using a novel method to predict binding to the conserved PxIxIT-docking surface on CN. Candidate sequences are validated for CN-binding in vitro, and in Aim 2 their parent proteins are tested for interaction with and/or dephosphorylation by CN in cultured animal cells. In Aim 3 we characterize the functions and binding mode of a newly discovered CN-binding motif, YLxxLF, which may identify a distinct class of CN- interacting proteins and provide avenues for selective disruption of CN signaling events. This systems- level analysis of CN signaling in humans will ultimately identify intersections with other regulatory networks that can be exploited therapeutically, such as providing strategies to ameliorate immunosuppressant side effects. Furthermore, this work will provide fundamental insights into how phosphatases achieve specificity and will create a critical new resource for researchers studying Ca2+ or phosphorylation-dependent regulation of protein function.